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Title:Nebulizer facilitated molding of freeform 3D printed sacrificial scaffolds for breast tumor microenvironment tissue engineering
Author(s):Gryka, Mark
Director of Research:Bhargava, Rohit
Doctoral Committee Chair(s):Bhargava, Rohit
Doctoral Committee Member(s):Harley, Brendan; Kong, Hyun-Joon; Underhill, Gregory
Department / Program:Bioengineering
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):sugar glass
3D printing
polymer film
epithelial model
vascular printing
tissue printing
Abstract:Tubular systems are essential for providing correct geometries in tissue engineered organs and models. Luminal structures are necessary for providing function in epithelial tissues including breast ducts, lungs, liver, and kidney. Cylindrical vessels are also necessary for expanding the size of model organs, delivering glucose and oxygen while removing CO2 and other waste products. Current methods for modeling organs, however, cannot span the range of resolutions necessary to create the complex tubular systems necessary in order to maintain these tissues. Model systems that can faithfully replicate fundamental micron scale features (ductal acini, capillaries, nephrons, etc.) do not easily scale to larger structures, while models of large structures fail to replicate intrinsic minute details. Sacrificial molding can be used to create tubular systems. Common molding techniques, however, lack spatial complexity for tissue engineering applications. Freeform 3D printing opens up sacrificial molding to the third dimension. For example, sugar glasses including isomalt offer facile, high resolution printing and simple removal of the sacrificial material but only offer short dwell times in aqueous media. This rapid dissolution makes it impossible to directly use this approach for precise molding of small features. Coating methods have been shown to have some efficacy as barriers to aqueous dissolution. A uniform coating is essential for downstream geometric and morphogenic events. In this work, we describe a method for nebulizing arbitrary solutions to the surface of isomalt 3D printed with smooth surfaces and >0.8 min/max cylindrical uniformity. Using this oscillating nebulizer system, we sought to test different types of barrier coatings for breast luminal modeling. Hydrophobic, reversible coatings were shown to be feasible for molding complex structures. This bio-orthogonal material could be completely washed out of molded hydrogels and had little interaction with cells due to its immiscibility with media. Using a combination of stimulated Raman scattering (SRS) microscopy and X-ray computed tomography, the coating was characterized to assess surface roughness and consistency. Colorimetric measurements of dissolution rates allowed optimization of sprayer parameters, yielding a decrease in dissolution rates by at least 4 orders of magnitude. High fidelity channels were ensured by surfactant treatment of the coating, which prevents bubbles from clinging to the surface. Spontaneous Raman scattering microspectroscopy and white light microscopy indicate cleared channels are free of octadecane following gentle flushing. The crystallinity and difficulty of removal limited the applicability of this material. Polymer coatings have a longer dwell time in the tissue mimic, but have the benefit of not requiring physical clearing of complex channels. In this work, we demonstrate a polymer-protein mixture and carefully characterize its properties as a nebulized coating material. Morphological properties were evaluated with a suite of analytical methods, including electron, optical, fluorescence, infrared (IR), and Raman microscopies. Functional characteristics of the material were also assessed, including dissolution kinetics, flux of fluorescent dextran after the isomalt scaffold was removed, and characteristics of cultured cells. The barrier significantly decreased the rate of scaffold dissolution with much thinner coatings than previously described. The new coating allowed generation of physiologically relevant multi-channel systems and cellularized lumens over a range of feature sizes as small as 50 μm. This material was then used to show the effects of print path on cell growth in the extra-luminal space. The freeform 3D printer was optimized for printing sacrificial nodules from 300 μm to 1000 μm on 200 μm luminal channels. These round features increase the cellular density changing the soluble factors around them, similar to solid tumors and vascular branching points. Mature luminal structures were grown outside of hydrogels, then incorporated in human umbilical vein endothelial cell laden hydrogels. Thus, the model lumens developed in this work provide a means of creating highly complex tissue mimics that promise the ability to expand tissue volume in a highly controlled fashion.
Issue Date:2020-11-18
Rights Information:Copyright 2020 Mark Gryka
Date Available in IDEALS:2021-03-05
Date Deposited:2020-12

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